CN116350803B - mRNA drug delivery system taking papillomavirus-like particles as carrier and preparation method thereof - Google Patents
mRNA drug delivery system taking papillomavirus-like particles as carrier and preparation method thereof Download PDFInfo
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Abstract
The application relates to the technical field of biological medicine, and particularly discloses an mRNA drug delivery system taking papillomavirus-like particles as a carrier and a preparation method thereof. By utilizing the self-assembly characteristic of main capsid protein L1 in papillomavirus-like particles, mRNA is added into papillomavirus-like particles dissociated by using DTT and EDTA, then after a stop buffer solution is added, the L1 protein is reassembled and polymerized into the papillomavirus-like particles containing mRNA inside, and the mRNA delivery system can be stably transported in vivo, so that mRNA completely enters cells to express corresponding protein, and the papillomavirus-like particles not only have good biocompatibility, non-immunogenicity and biodegradability, but also can act in an oral mode, and the expression quantity of target protein is high.
Description
Technical Field
The application relates to the technical field of biological medicine, in particular to an mRNA drug delivery system taking papillomavirus-like particles as carriers and a preparation method thereof.
Background
mRNA is a single-stranded ribonucleic acid which is transcribed from one strand of DNA as a template, carries genetic information and can guide protein synthesis, and has the size of 300-5000 kDa and the length of 1-15 kb. Compared with DNA, mRNA does not enter the nucleus, and protein synthesis and functional activity can be completed in cytoplasm, and DNA needs to enter the nucleus first and then be transcribed into mRNA, so that the efficiency of DNA is lower than that of mRNA. In addition, mRNA can not be inserted into genome, but only transiently expresses coding protein, so that the risk of mutation caused by insertion into genome is reduced, the safety is better, the mRNA is easy to synthesize through an in vitro transcription process, the process is simple, the industrial production cost is relatively low, the mRNA can be rapidly applied to various therapies, and the application potential is extremely wide.
mRNA can theoretically express any protein, and plays a role in treating various diseases by expressing functional proteins in vivo, but the single-chain structure of mRNA is unstable and easy to degrade, and the mRNA carries negative charges, so that delivery through cell membranes with the same negative charges on the surface is also a difficult problem. Thus, mRNA is delivered to the cell interior with a total of two barriers: first, enzymatic degradation during delivery; secondly, membrane barriers caused by electrostatic repulsion, so that in the practical application process, special modification of mRNA and matching with a delivery system are required to realize intracellular expression of mRNA. Lipid nanoparticles (Lipid nanoparticle, LNP) are the most popular delivery technology at present, and besides mRNA bearing LNP carriers containing negatively charged mRNA, there are four other components, namely ionizable cationic phospholipids (ionizable lipids), neutral helper phospholipids, cholesterol and polyethylene glycol modified phospholipids (PEGylated lipids), which can protect mRNA molecules from extracellular degradation or promote fusion of mRNA molecules with cell membranes to enhance transfection. The most commonly used means of mRNA vaccine delivery today are, besides Lipid Nanoparticles (LNP), cationic lipid complexes (LPX), lipid polyplexes (LPP), polymer nanoparticles (Polymer nanoparticles, PNP), inorganic nanoparticles (Inorganic nanoparticles, INP), and cationic nanoemulsions (Cationic nanoemulsion, CNE), etc.
However, the above-described existing mRNA delivery system has the following problems: firstly, these nanoparticies are partially composed of exogenous components, LNPs activate different inflammatory pathways, which lead to the production of inflammatory cytokines such as IL-1 and IL-6, which can initiate and maintain local and systemic inflammation and side effects, most of which are derived from LNP components such as PEG, ionized lipids, etc., which lead to innate or adaptive immunity against the delivery system; secondly, the preparation process is complex, the traditional preparation method of mRNA/LNP is that two phases are mixed and then the particle size is controlled by a homogenization technology (a high-pressure homogenizer and high-pressure microjet) or an extrusion technology (a polycarbonate membrane filter), and the preparation difficulty is high due to high process requirements; thirdly, the preparations are all required to be injected intramuscularly, so that the use is inconvenient; fourth, lipid nanoparticles require low temperature storage and are therefore very unstable.
In summary, there is a need for an mRNA delivery system that is biocompatible, easy to prepare, orally-available, and easy to preserve.
Disclosure of Invention
In order to solve the problems, an mRNA delivery system using papillomavirus-like particles as a carrier and a preparation method thereof are provided, and the mRNA-loaded papillomavirus-like nanoparticles have good stability, can ensure that the mRNA-loaded papillomavirus-like particles are completely transported in vivo, have good biocompatibility, non-immunogenicity and biodegradability, and have higher expression level of target proteins compared with the prior art.
According to one aspect of the present application, there is provided a method for preparing an mRNA delivery system using papillomavirus-like particles as a carrier, the method comprising the steps of: the mRNA is loaded into the interior of papillomavirus-like particles to obtain the mRNA delivery system.
Optionally, the process specifically includes the following steps:
1) Mixing the papillomavirus-like particles with a dissociation buffer solution and dissociating to obtain a solution A;
2) Adding mRNA into the solution A, mixing to obtain solution B;
3) And mixing the solution B with a stop buffer solution, and then performing reassembly to obtain a solution C containing the mRNA drug delivery system.
Optionally, the dissociation buffer comprises ethylene glycol di (β -aminoethyl ether) tetraacetic acid (EGTA), dithiothreitol (DTT), sodium chloride (NaCl), and Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl); preferably, in the dissociation buffer, the concentration of ethylene glycol di (beta-aminoethyl ether) tetraacetic acid (EGTA) is 20+/-1 mM, the concentration of Dithiothreitol (DTT) is 40+/-2 mM, the concentration of sodium chloride (NaCl) is 300+/-10 mM, and the concentration of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) is 100+/-5 mM; preferably, the concentration of ethylene glycol di (beta-aminoethyl ether) tetraacetic acid (EGTA) is 20mM, the concentration of Dithiothreitol (DTT) is 40mM, the concentration of sodium chloride (NaCl) is 300mM, and the concentration of Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) is 100mM.
Preferably, the dissociation time is 50-70 min, preferably 60min.
Preferably, the volume ratio of papillomavirus-like particles to dissociation buffer is 1:0.9 to 1.1, preferably 1:1.
optionally, the stop buffer comprises calcium chloride (CaCl) 2 ) And dimethyl sulfoxide (DMSO); preferably, in the stop buffer, the calcium chloride (CaCl 2 ) A concentration of 25+ -1 mM, and a concentration of 20+ -1 vol.% of dimethyl sulfoxide (DMSO); preferably, in the stop buffer, the calcium chloride (CaCl 2 ) The concentration was 25mM, and the concentration of dimethyl sulfoxide (DMSO) was 20vol%.
Preferably, the temperature of the recombinant assembly is 2-6 ℃, preferably 4 ℃.
Optionally, step 3) is followed by step 4) for purifying the mRNA delivery system:
4) Firstly adding the solution C onto a sucrose/PBS layer, collecting a precipitate after primary centrifugation, then adding PBS for re-suspension, adding the obtained re-suspension into a CsCl solution, collecting a secondary precipitate after secondary centrifugation, and finally filtering and embedding by using a filter membrane to obtain the purified mRNA drug delivery system;
preferably, the primary centrifugation condition is 27000r/min and centrifugation is carried out at 4 ℃ for 3 hours; preferably, the secondary centrifugation condition is 35000r/min and centrifugation is carried out at 4 ℃ for 20h.
Alternatively, the papillomavirus-like particle is comprised of papillomavirus major capsid protein L1.
Alternatively, the mRNA is a modified mRNA having 5 'caps, 5' and 3 '-untranslated regions and a 3' -polyA tail.
Preferably, the modification further comprises replacing conventional nucleotides with chemically modified nucleotides, replacing cytidine with 5-methylcytidine (m 5C), and/or replacing uridine with 5-methyluridine (m 5U), and/or replacing adenosine with N1-methyladenosine (m 1A), N6-methyladenosine (m 6A), 2-thiouridine (s 2U), 5-methoxyuridine (5 moU), pseudouridine (ψ), or N1-methylpseudouridine (m 1 ψ).
Alternatively, the mRNA may encode one or more of an antibody protein, a tumor antigen, and a pathogen protein.
According to another aspect of the present application, there is provided an mRNA delivery system using papillomavirus-like particles as a carrier, characterized in that the mRNA delivery system is prepared according to any one of the preparation methods described above.
Alternatively, the mRNA delivery system is administered by injection or orally; preferably, the mRNA delivery system is used orally.
The beneficial effects of the application include, but are not limited to:
1. the preparation method of the mRNA drug delivery system taking the papillomavirus-like particles as the carrier provided by the application prepares and obtains the mRNA drug delivery system taking the papillomavirus-like particles as the carrier, provides a new thought and method for mRNA delivery, and has simple preparation method and process compared with Lipid Nano Particles (LNP) and a polymer mode, and the oral mRNA drug delivery system has non-immunogenicity and is not easy to cause potential adverse events such as anaphylactic reaction and the like.
2. The mRNA drug delivery system taking papillomavirus-like particles as the carrier has good stability and biocompatibility, can ensure that the drug delivery system can be completely transported in vivo, has the characteristic of biodegradability, and is an ideal carrier for easily degradable drugs.
3. The mRNA drug delivery system using papilloma virus-like particles as a carrier provided by the application enters the intestinal tract through an oral way, immune cells can completely enter cells through combination with papilloma virus receptors in a pinocytosis way and express corresponding proteins, the expressed proteins play a role in preventing and treating diseases for various antibodies, immune regulatory factors and the like, and compared with the existing plasmid delivery technical scheme, the target protein expression quantity of the mRNA drug delivery system is higher.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:
fig. 1: the structure of recombinant expression plasmid pRP [ Exp ] -Puro-EF1A > { Igk leader/EGFP };
fig. 2: the structure of the in vitro transcription vector pUC19K-T7pA110-UTRM-Ig kappa-sig/EGFP is schematically shown;
fig. 3: schematic of particles under transmission electron microscope: a, bovine papilloma virus-like particle BPV1 particles; b, human papillomavirus HPV16 particles (33000X);
fig. 4: agarose gel electrophoresis of mRNA extraction (10 different samples 1-10);
fig. 5: detecting a standard curve of EGFP concentration;
fig. 6: mRNA-loaded VLPs infect CaCo-2 cell lines to express EGFP levels;
fig. 7: mice perfused with in vivo EGFP levels after loading mRNA VLPs.
Detailed Description
The present application is described in detail below with reference to examples, but the present application is not limited to these examples.
Unless otherwise indicated, the starting materials and reagents in the examples of the application were all purchased commercially.
In the mRNA delivery process, since naked mRNA is taken up by cells through the scavenger receptor mediated endocytic pathway and accumulated in endosomes, so that the efficiency of the naked mRNA in vivo transportation and penetration of cell membranes into cytoplasm is extremely low, the in vivo normal cell uptake of mRNA is very low, and only immature dendritic cells can take up and effectively accumulate mRNA through the megakaryocyte pathway, so that a more efficient delivery mode is required for the wide application of therapeutic mRNA.
In recent decades, mRNA formulation technology has evolved, such as lipid nanoparticles and polymers, to efficiently deliver mRNA into most types of cells, where mRNA-encapsulated nanoparticles are taken up by endocytosis and then released from the endosome, where the ribozyme will initiate translation and produce various proteins including secreted, transmembrane, intracellular, and mitochondrial proteins, etc. However, such formulation techniques require the use of large amounts of chemicals, such as ionizable cationic phospholipids, neutral helper phospholipids, cholesterol, PEG-modified phospholipids, and the like, not only are complex processes, but also are prone to adverse events such as allergic reactions.
mRNA is used as a potential candidate drug, but the naked mRNA has the stability problem of being easy to degrade in vitro, short in vivo half-life and poor in patentability when used as a drug. In order to improve mRNA stability, various chemical modifications such as 5' -capping, polyadenylation (A) tail, 5' -and 3' -UTR (untranslated region) and codon optimization of coding region are required to be performed on mRNA structure, so that the problem of mRNA instability can be solved. Methylation modification (such as m7G and the like) of the 5' cap, substitution of a phosphorothioate contained in a triphosphate bridge and the like can promote combination of mRNA and ribosome, effectively seal the 5' end of the RNA, protect 5' exonuclease degradation of the mRNA and enhance the stability of the mRNA; polyadenylic acid (polyA) at the 3' end of mRNA plays a key role in regulating mRNA stability and translation efficiency, while also reducing uridine exposure to reduce immunogenicity; the 5 '-and 3' -UTRs in the mRNA contain specific regulatory sequence elements that regulate translation and stability of the mRNA; the half-life of the mRNA can be optimized by introducing a stability element in the UTR.
Optimization of codons may also play a further regulatory role, e.g., synonymous codon substitutions may have a significant impact on protein expression, folding and cellular function, e.g., mRNA secondary structure may regulate protein expression by altering half-life of mRNA translation, and chemically modified nucleotides may stabilize mRNA spatial structure to increase protein expression levels; at the same time, optimization of GC-rich codons and minimization of U content, or several nucleotide chemical modification strategies to reduce immunogenicity without affecting its translational properties, such as replacement of natural uridine with 5-methyluridine (m 5U) and pseudouridine (ψ) can reduce mRNA immunogenicity, enhance transcript stability, and also improve translation efficiency, can be chosen.
After improving the stability of mRNA, there is still an inefficiency in the transport of naked mRNA in vivo and across the cell membrane into the cytoplasm. Studies have demonstrated that naked mRNA is taken up by cells via scavenger receptor mediated endocytosis and accumulated in endosomes (endosomes), that normal cells take up mRNA in vivo inefficiently, and that only immature dendritic cells can take up and effectively accumulate mRNA via the macropolytics pathway, so that the broad use of therapeutic mRNA requires a more efficient delivery means to be able to transport the injected mRNA intact from the injection site to the corresponding target moiety.
In recent decades, mRNA formulation technology has evolved, for example, using Lipid Nanoparticles (LNP) and polymers, etc., to efficiently deliver mRNA into most types of cells, where mRNA-encapsulating nanoparticles are taken up by endocytosis and then released from the endosome, where the ribozyme will initiate translation and produce various proteins including secreted, transmembrane, intracellular, and mitochondrial proteins, etc. However, such preparation technology has a large amount of chemicals, such as ionizable cationic phospholipids, neutral auxiliary phospholipids, cholesterol, PEG modified phospholipids and the like, and is complex in process and easy to cause adverse events such as anaphylactic reaction and the like.
In searching for mRNA delivery systems, the inventors have unexpectedly found that human papillomavirus-like particles can encapsulate non-replicating, modified mRNA therein, have transport stability, transport mRNA expresses mRNA-encoded proteins in cells, expresses target gene-encoded proteins in vivo, and has significant advantages over delivery of DNA (e.g., expression plasmids). The inventor discovers that by utilizing the technical advantages of mRNA modification and adding the virus-like particles with better degradability, mRNA can be expressed in vivo more permanently and the biological effect is more obvious. In the application, the inventor takes papillomavirus-like particles as the oral administration drug delivery system of the mRNA, not only provides a new thought and mode for mRNA delivery, but also has the advantages of high stability and biocompatibility compared with the prior art adopting a Lipid Nanoparticle (LNP) and polymer mode, and has the characteristics of non-immunogenicity and biodegradability, so that the safety of the use is higher, the difficult problem of mRNA delivery can be solved, and the papillomavirus-like particles prepared by the technology can be expressed by genetic engineering.
In addition, the mRNA delivery system may also be orally administered. There is currently no nucleic acid drug or nucleic acid vaccine available for oral administration in the world, mainly because the intestinal system contains complex and large amounts of enzyme systems, which rapidly degrade nucleic acids, and the intestinal parietal cells have difficulty in taking nucleic acids and expressing functional proteins. The inventors have found that papillomavirus L1 protein can encapsulate mRNA well, and can take advantage of the dual characteristics of digestive enzyme resistance and intestinal wall-friendly epithelial cells of compact pseudovirus particles formed by papillomavirus L1 protein, so that the carried mRNA passes through the intestinal tract, is combined with the intestinal epithelial cells, is delivered into cells, and expresses secreted or membrane-combined functional protein (according to mRNA design) on the intestinal wall, thereby playing the role of treatment or vaccine of the functional protein.
Papillomavirus L1 proteins, or formed virus-like particles (VLPs) or pseudovirions, bind to intestinal epithelial cells and are mediated by uptake of heparin sulfate polysaccharide proteins (heparan sulfate proteoglycan) through specific spatial conformations (literature: knapp, M.et al Surface-exposed amino acid residues of HPV L1 protein mediating interaction with cell Surface heparan sulfate. J. Biol. Chem. 2007,282,27913-27922). The mechanism by which pseudoviruses formed by papilloma L1 proteins enter cells is generally thought to be via clathrin (clathrin) and microcapsule (canelar) -mediated pinocytosis (Bousarghin, L.AT, et al Human papillomavirus types, 31, and 58 use different endocytosis pathways to enter cells. J virol 2003, 77:3846-50; laniosz VKA et al Bovine papillomavirus type 1: from clathrin to canelal J virol 2008). Intracellular mRNA is translated into the corresponding functional protein by ribosomes and released into the surrounding tissue or into the blood circulation.
Human papilloma virus (human Papilloma virus, HPV), which belongs to a group of small DNA non-cytolytic viruses, has no envelope and has a diameter of 52-55 nm. Papillomavirus late coding region L1 and L2 genes encode the major (L1) and minor (L2) capsid proteins of HPV, respectively. The L1 proteins have a self-assembly function in vitro, 5L 1 proteins are polymerized into one pentamer, and then 72 pentamers are assembled into one virus-like particle (VLPs) automatically, so that one VLP contains 360L 1 proteins. The main capsid protein L1 has a molecular weight of 56kDa and consists of 505 amino acids, accounting for approximately 80% of the total viral structural protein. HPV has epitheliophilic properties (tropic), and L1 protein can bind with receptors such as heparan sulfate proteoglycan ((heparan sulfate proteoglycans, HSPG) on the surface of epithelial cells of skin, mucosa and the like and can be ingested by endocytosis.
VLPs may be obtained by genetic engineering, using HPV16 virus-like particle (VLPs) preparation as an example: constructing HPV16L1 gene on pBacPAK8, and obtaining recombinant virus containing L1 gene by homologous recombination with linearized baculovirus in sf9 insect cells; HPV16 virus-like particles (VLPs) can be assembled in the nuclei of insect cells by infection of sf9 insect cells with recombinant virus, and VLPs can be purified by ultracentrifugation with 30% CsCl; VLPs may also be commercially available.
The VLPs can be dissociated into L1 protein in the presence of DTT and EDTA, mRNA which has been transcribed and synthesized in vitro is added to the dissociated L1 protein, and after the DTT and EDTA are removed, the concentration of calcium ions is increased so that the dissociated L1 protein can be repolymerized into VLPs, wherein a part of the VLPs coated with the mRNA is called virus-like particles or pseudo-virus particles, and the part of the VLPs coated with the mRNA is the desired mRNA delivery system. The inventors found that the use of DTT and EGTA-containing buffer to deagglomerate virus-like particles, then mixing with mRNA, working well when the time after mixing is 60min, then re-using increasing concentrations of CaCl 2 Standing at 4deg.C for 12 hr to obtain complete pseudo virus particle, i.e. mRNA delivery system.
One of the objectives of the present application is to develop a papillomavirus-like particle capable of loading mRNA, from which self-produced or commercially available papillomavirus L1 proteins dissociate after reduction, and from which mRNA can be loaded stepwise to self-assemble particles similar in appearance to natural papillomaviruses. It is another object of the present application to produce a stable, biocompatible nanoparticle that is different from artificial LNP. The inventor researches find that the papillomavirus-like nano particles loaded with mRNA have certain stability, so that the papillomavirus-like particles loaded with mRNA can be completely transported in vivo, and the biocompatibility, the non-immunogenicity and the biodegradability of the papillomavirus-like nano particles are ensured, so that the papillomavirus-like nano particles become ideal carriers for easily degradable medicines.
The papillomavirus-like particle can be stably loaded with non-replicative modified mRNA (messenger ribonucleic acid) with various proteins for coding and treating diseases, the papillomavirus-like nanoparticle is delivered into the intestinal tract by injection and oral administration, and the modified mRNA in the papillomavirus-like particle completely enters cells and expresses corresponding proteins in a pinocytosis mode by combining with a papillomavirus receptor, and the expressed proteins play a role in preventing and treating diseases for various antibodies, immune regulation factors and the like. The gastrointestinal tract contains 70% -80% of the immune cells of the human immune system, and the gut-associated lymphoid tissue (gat-associated lymphoid tissue, GALT) is the main part of the immunity that constitutes the gut, mainly including Peyer's patch PP, independent lymphoid follicles, mesenteric lymph nodes, and lymphocytes within the lamina propria, etc. located in the wall of the small intestine. Therefore, by orally delivering mRNA expressing immune related proteins, the mRNA delivery system can enter cells more efficiently, thereby realizing the expression of target mRNA and achieving better therapeutic effect.
In order to accomplish the object of the present application, the present application adopts the technical scheme of the following embodiments. In order to facilitate the detection of whether mRNA is successfully expressed, the feasibility and effect of the application are examined by using Enhanced Green Fluorescent Protein (EGFP) as a tool.
EXAMPLE 1 preparation of mRNA delivery System
Preparation of mRNA using an in vitro transcription System (IVT-mRNA)
(1) Construction and preparation of recombinant expression plasmid
The expression plasmid pRP [ Exp ] -Puro-EF1A > { Igk leader/EGFP } (shown in FIG. 1) was constructed by the Kyoho biotechnology (Guangzhou) Co., ltd.) to enhance the green fluorescent protein EGFP as a tool protein for control studies. Constructing a vector by using Gateway technology, comprising two steps of reaction: the first step of BP reaction is to construct an entry cloning vector containing a target sequence; in the second step, the LR reaction constructs a recombinant expression vector containing a target sequence, and in the Gateway technology, the important recombination sites are: attB, attP, attL and attR. The method comprises the following specific steps: amplifying target genes, and designing a primer sequence according to a BP reaction principle: attB site + specific primer sequences (underlined).
pD-PF1, as shown in SEQ ID NO. 1:
GGGGACAAGTTTGTACAAAAAAGCAGGCTGCCACCATGGAGACAGACACACTCCTGC；
pD-PR1 as shown in SEQ ID NO. 2:
GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTGTACAGCTCGTCCATGCC。
and (3) performing gel cutting recovery on the target fragment, performing BP reaction to construct an intermediate vector pDOWN- { Igk leader/EGFP }, performing sequencing identification on the intermediate vector, performing LR reaction, recombining the vector pUp-EF1A, pDown- { Igk leader/EGFP }, pRP [ Exp ] -Puro, transforming, performing final vector positive cloning identification, and finally constructing and obtaining the pRP [ Exp ] -Puro-EF1A > { Igk leader/EGFP } vector.
(2) In vitro transcription of mRNA, purification and detection
mRNA was synthesized by the Sophora alopecuroides (Guangzhou) Inc. Firstly, constructing an in vitro transcription vector pUC19K-T7pA 110-UTRM-Igkappa-sig/EGFP (shown in figure 2), wherein a construction element comprises T7 promoter-5 'UTR-ORF-3' UTR-polyA, and a target gene is EGFP, and the EGFP is preceded by an Igkappa signal peptide sequence so as to facilitate the EGFP to secrete cells; preparing the above vector, linearizing the purified vector with single enzyme, selecting mMESSAGE mMACHINE T7 Ultra Kit (Invitrogen ™), and performing in vitro transcription according to instruction requirementCapping and poly A reactions, 15mM UTP of kit T7 2X NTP/ARCA replaced with N1-Methylpseudouridine-5 '-triphosphates (N1 pseudouridine-5' -Triphosphate, trilink Biotech); the synthesized modified mRNA was recovered and purified by using the NucAway ™ Spin ColumnsMEGAclear ™ Kit, and the recovered mRNA was purified according to 1:100 volumes were diluted and the A260 and A280 values were determined using an ultraviolet spectrophotometer, based on 1 OD of synthesized RNA 260 Quantitative =33 μg, and OD was calculated 260 /OD 280 Ratio of the two.
The Igkappa-sig/EGFP element size in the vector is 783bp, the molecular weight is about 258 kDa, and the average OD260/280 ratio obtained by an ultraviolet-visible spectrophotometer is 2.02, which indicates that the purity of the synthesized mRNA is high.
Assembly and purification of mRNA-encapsulated papillomavirus-like nanoparticles
Firstly, mixing the outer shell of human or bovine papillomavirus (recombinant HPV16L1 protein, product number ab119880; recombinant BPV L1 protein, product number CBS-V554) with dissociation buffer solution according to the volume of 1:1, and culturing for 60 minutes at room temperature, wherein the dissociation buffer solution is: ethylene glycol di (. Beta. -aminoethyl ether) tetraacetic acid (EGTA) 20mM, dithiothreitol (DTT) 40mM, sodium chloride (NaCl) 300mM, tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) (pH 8.0) 100mM; then 1/10 volume of modified mRNA was added at a concentration of 0.51. Mu.g/. Mu.l; gradually adding a stop buffer solution, wherein the stop buffer solution is as follows: calcium chloride (CaCl) 2 ) 25mM, dimethyl sulfoxide (DMSO) 20vol%, and the mixed solution was left at 4℃overnight.
After self-assembling into virus-like particles (VLPs), slowly adding the self-assembled VLPs mixed solution on a 30% (w/w) sucrose/PBS layer, centrifuging at 27000rpm and 4 ℃ for 3 hours, and taking a precipitate (small yellow circular plaque-like substance) and adding 1mLPBS for resuspension; the heavy suspension was slowly added to 10mL of 29% CsCl solution along the vessel wall, and the precipitate was collected after centrifugation at 35000r/min at 4℃for 20h, and the chimeric VLPs sample was filtered through a 0.22 μm filter to obtain the desired mRNA delivery system.
The particle size of the virus-like particles was measured by Malvern Zetasizer Nano ZS nm particle size potentiometric analyzer, zetasizer Software software analysis data: dropping the sample on a carbon-sprayed copper net, standing for 5min, then dropping 2% potassium phosphotungstate for dyeing, standing for 5min, naturally semidrying, fixing, setting the magnification of a transmission electron microscope to 50000 times, and observing the particle shape of the sample at 80 kV. As shown in FIG. 3, the in vitro assembled VLPs were subjected to dynamic light scattering particle size analysis and transmission electron microscopy, and a large number of spherical particles having a morphology and a diameter of 50 to 55nm were observed, and the particle sizes were uniform, indicating that the in vitro self-assembled VLPs were loaded.
Experimental example 1 mRNA encapsulation efficiency detection of EGFP expression
mRNA-loaded papillomavirus-like particles were prepared as in example 1, peripheral RNA not encapsulated was removed by digestion with high concentration RNase, washed with RNase-free PBS buffer, and then disaggregated with the dissociation buffer of example 1 (dissociation buffer: ethylene glycol di (. Beta. -aminoether) tetraacetic acid (EGTA) 20mM, dithiothreitol (DTT) 40mM, sodium chloride (NaCl) 300mM, tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) (pH 8.0) 100 mM), and low concentration of RNase inhibitor was added.
After confirming nanoparticle deagglomeration and protein quantification by electron microscopy or electrophoresis, mRNA qualitative and quantitative analysis was performed on Agilent 2100 Bioanalyzer or Agilent Bioanalyzer system or on the same using RNA extraction reagents, confirming that complete mRNA was encapsulated into virus-like particles. The results showed that mRNA was detectable in the mRNA papillomavirus-like particles loaded, and that the amount of encapsulated mRNA was 200-500ng mRNA/mg protein in the mRNA papillomavirus-like particles. The encapsulation rate is calculated by the following steps: the amount of encapsulated mRNA/total mRNA added was 100% and the average encapsulation efficiency could reach 72.2±8.5%.
Experimental example 2 papillomavirus-like particles coating mRNA resistant to nuclease digestion
Human or bovine papilloma virus-like particles act with nuclease Benzonase (Sigma-Aldrich, E8263, > 250 units/. Mu.L, final concentration 250U/mL) for different times, RNA is extracted by using a viral RNA extraction kit (QIAamp Viral RNA Mini Kit) produced by QIAGEN company, and mRNA is quantified by using a micro ultraviolet spectrophotometer; and then, performing EB-stained agar gel electrophoresis, and observing the integrity of the modified mRNA wrapped by the pseudo virus particles. The results are shown in FIG. 4 below, and after various time periods of nucleic acid treatment, it can be seen that mRNA is not degraded and remains intact.
Test example 1 optimization procedure of preparation conditions
Firstly, mixing the outer shell of human or bovine papilloma virus (recombinant HPV16L1 protein, product No. ab119880; recombinant BPV L1 protein, product No. CBS-V554) with dissociation buffer solution according to the volume of 1:1, and in order to prevent mRNA from being degraded in vitro, the encapsulation needs to be completed in the shortest time possible, and the influence of time on the encapsulation efficiency needs to be considered, so that the combined action time of the recombinant encapsulation process in the step 3) is examined, and the specific settings are 30min, 40min, 50min and 60min. The inventors found that the encapsulation efficiency was not high at 30min, whereas mRNA was likely degraded at 60min (encapsulation efficiency measurement method see experimental example 1), so that the co-action was selected for 40min.
The inventors also conducted screening studies at room temperature on the final concentration (10 mM, 20mM, 40mM, 50 mM) of Dithiothreitol (DTT) in the dissociation buffer, with average encapsulation efficiencies of 49.7%, 76.4%, 71.3% and 69.4%, respectively. The dissociation buffer was finally determined to be: ethylene glycol di (. Beta. -aminoethyl ether) tetraacetic acid (EGTA) 20mM, dithiothreitol (DTT) 20mM, sodium chloride (NaCl) 300mM, tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) (pH 8.0) 100mM; then 1/10 volume of modified mRNA was added at a concentration of 0.51. Mu.g/. Mu.l; the stop buffer was then added gradually (3 times, one third volume each) and the final stop buffer concentration was determined to be: calcium chloride (CaCl) 2 ) 10mM, dimethyl sulfoxide (DMSO) 20vol%. The inventors also examined the time of overnight treatment, and the mixed solution was left to stand at 4℃overnight for 12 hours, and showed that the pseudo-virus particles prepared at 12 hours of treatment of the mixed solution were more complete as compared with 2 hours of treatment.
EXAMPLE 2 expression of the protein of interest after in vitro transfection of cells with mRNA papillomaVirus-like particles
Inoculating human colon cancer CaCo-2 cell strain (ATCC HTB-37) into 96-well plate, 1X10 4 Cells CaCo-2/well overnight, after 80% cell fusion, appropriate amount of serum-free medium was added and blotted off, 3 replicate wells per group. The following groups are set: (1) Test group, 10. Mu.g of mRNA-loaded papillomavirus particles per well, 1mL of culture solutionSystem (serum-free); (2) plasmid control: 10 μg of expression plasmid-loaded papillomavirus particles (volume as above) were added per well, 1mL of culture solution system; (3) Liposome-mediated transfection group: mu.g of mRNA per well was mixed with 10. Mu.L/Kong Zhi plastid (Lipofectamine 3000), left at room temperature for 20min, and added to the cell culture system. A control group of papillomavirus-like particles and a naked mRNA group were additionally provided without nucleic acid. 3 duplicate wells/group, cells of the above group were cultured for 4h at 37℃and then added with 2mL of complete culture solution, followed by further culture for 48h, detection of supernatant by aspiration, and detection by ELISA after cryopreservation (Kit GFP ELISA Kit, ab 171581). The concentration of EGFP was measured according to the standard curve of EGFP concentration and OD value in FIG. 5, and the result showed that the expression level of the target protein was highest after the pseudo-viral particle loaded with mRNA was infected with the cell (EGFP concentration results are shown in Table 1 and FIG. 6 below).
As can be seen from the results in Table 1, the mRNA delivery system provided by the present application has the highest expression level of the final target protein compared to the plasmid loading method and the conventional liposome-mediated delivery system.
EXAMPLE 3 expression of mRNA-loaded papillomavirus-like particles in animals
KM mice, 3 groups: a control group of human papillomavirus-like particles, a group of mRNA-loaded papillomavirus-like particles, and a treated group of expression plasmid-loaded human papillomavirus-like particles. 4 animals/group, male and female, 3 groups marked with colors (head, back and tail), and different cages were placed, and the mice were orally administrated 1 time, the volumes were 200 μl/mouse, and blood was taken and serum was isolated at 2 time points 24h and 72h after the administration of pseudovirions (L1 protein), and the mRNA-encoded EGFP level in the serum was detected.
The results showed that higher levels of EGFP could be detected in the serum of the pseudovirion group mice loaded with mRNA, which on average reached 0.624pg/mL, whereas higher levels of EGFP could be detected in the serum of the pseudovirion group mice loaded with expression plasmid, which was only 0.193pg/mL, indicating that mRNA delivery was more efficient with HPV pseudovirions and higher levels of the protein of interest could be expressed in vivo (results see Table 2 and FIG. 7 below).
As can be seen from the results of Table 2, the mRNA delivery system provided by the present application can express higher levels of the protein of interest in vivo than conventional means of delivering plasmids.
In summary, the mRNA delivery system provided by the application not only improves the effect of expressing the target protein, but also can play a role in oral administration, and the preparation method of the mRNA delivery system is simple, compared with the existing delivery system, the mRNA delivery system has higher safety, the expression amount of the target protein is higher, and the reasons for the results are probably as follows: for the scheme of loading the human papillomavirus-like particles for expressing plasmids, the plasmids carry DNA sequences, and the target proteins can be expressed after mRNA is transcribed after entering the cell nucleus when the plasmids play a role, so that the expression quantity of the target proteins is not only dependent on the fact that the plasmids are introduced into the cell cytoplasm, but also the influence factors of the plasmids entering the cell nucleus are considered, in the scheme of the application, mRNA fragments can be translated after entering the cell cytoplasm so as to express the target proteins, and the expression quantity is higher; for a liposome-mediated mRNA delivery system, the mRNA serving as a carrier is negatively charged and can have a strong connection effect with the liposome serving as a carrier, even if the liposome is successfully fused with a cell membrane, the mRNA does not enter the cytoplasm completely, but is easily adhered to the cell membrane partially and then degraded by the cell, and cannot participate in the expression and translation process of a target protein, so that the expression quantity of the target protein is relatively low, the liposome has relatively definite cytotoxicity, and the cationic liposome alone cannot be used in a human body as a delivery body.
According to the scheme of the application, VLPs of HPV are used for wrapping mRNA to realize that the mRNA is delivered into cells for expressing target proteins, and the expression quantity can be greatly improved compared with the prior art through an oral administration mode, and the preparation method of the mRNA drug delivery system in the scheme not only enables the VLPs to wrap a large amount of mRNA, but also can resist enzymatic degradation of the digestive tract through an oral administration mode, and the mRNA is delivered into cells for high-efficiency expression after being combined with intestinal epithelium, so that the administration mode is convenient and quick, and the administration difficulty is obviously reduced.
The above description is only an example of the present application, and the scope of the present application is not limited to the specific examples, but is defined by the claims of the present application. Various modifications and variations of the present application will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application should be included in the protection scope of the present application.
Claims (5)
1. An application of an mRNA delivery system taking papillomavirus-like particles as a carrier in preparing an oral mRNA drug, which is characterized in that the preparation method of the mRNA delivery system comprises the following steps: loading mRNA inside papillomavirus-like particles consisting of papillomavirus major capsid protein L1, obtaining said mRNA delivery system;
the process specifically comprises the following steps:
1) Mixing the papillomavirus-like particles with a dissociation buffer solution and dissociating to obtain a solution A;
2) Adding mRNA into the solution A, mixing to obtain solution B;
3) Mixing the solution B with a stop buffer solution, and then reassembling to obtain a solution C containing the mRNA drug delivery system;
the dissociation buffer solution comprises ethylene glycol di (beta-aminoethyl ether) tetraacetic acid (EGTA), dithiothreitol (DTT), sodium chloride (NaCl) and Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), wherein the concentration of the ethylene glycol di (beta-aminoethyl ether) tetraacetic acid (EGTA) is 20+/-1 mM, the concentration of the Dithiothreitol (DTT) is 40+/-2 mM, the concentration of the sodium chloride (NaCl) is 300+/-10 mM, the concentration of the Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) is 100+/-5 mM, and the dissociation time is 50-70 min;
the stop buffer comprises calcium chloride (CaCl) 2 ) And dimethyl sulfoxide (DMSO), the calcium chloride (CaCl) 2 ) The concentration is 25+/-1 mM, the concentration of dimethyl sulfoxide (DMSO) is 20+/-1 vol%, and the temperature of the recombinant assembly is 2-6 ℃;
the papillomavirus is HPV16.
2. The use of claim 1, wherein the mRNA is a modified mRNA having 5 'caps, 5' and 3 '-untranslated regions, and a 3' -polyA tail; the modification also includes using chemically modified nucleotides instead of conventional nucleotides, 5-methylcytidine (m 5C) instead of cytidine, and/or 5-methyluridine (m 5U) instead of uridine, and/or N1-methyladenosine (m 1A), N6-methyladenosine (m 6A), 2-thiouridine (s 2U), 5-methoxyuridine (5 moU), pseudouridine (ψ) or N1-methylpseudouridine (m 1 ψ) instead of adenosine.
3. The use of claim 1, wherein the mRNA may encode one or more of an antibody protein, a tumor antigen, and a pathogen protein.
4. The use according to claim 1, wherein the volume ratio of papillomavirus-like particles to dissociation buffer is 1:0.9 to 1.1.
5. The use according to claim 1, characterized in that said step 3) is followed by a step 4) for purifying said mRNA delivery system:
4) Adding the solution C onto a sucrose/PBS layer, collecting a precipitate after primary centrifugation, adding PBS for re-suspension, adding the obtained re-suspension into a CsCl solution, collecting a secondary precipitate after secondary centrifugation, and finally filtering and embedding by using a filter membrane to obtain the purified mRNA drug delivery system.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020084620A (en) * | 2001-05-03 | 2002-11-09 | 오유경 | Gene delivery system containing a desired gene in virus-like particles |
| US6599739B1 (en) * | 1996-07-17 | 2003-07-29 | The United States Of America As Represented By The Department Of Health & Human Services | Infectious papillomavirus pseudoviral particles |
| CN101487009A (en) * | 2008-01-15 | 2009-07-22 | 上海泽润生物科技有限公司 | Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system |
| CN115960178A (en) * | 2022-12-28 | 2023-04-14 | 北京康乐卫士生物技术股份有限公司 | Expression of human papilloma virus HPV59L1 protein, virus-like particle and preparation method thereof |
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| GB0007231D0 (en) * | 2000-03-24 | 2000-05-17 | Chiron Spa | Modified rna for gene delivery |
| CN107252489A (en) * | 2009-04-13 | 2017-10-17 | 法国健康和医学研究院 | HPV particles and application thereof |
| US20200010850A1 (en) * | 2017-02-17 | 2020-01-09 | The USA, as represeented by the Secretary, Dept. of Health and Human Services | Efficient cell free production of papillomavirus gene transfer vectors |
| WO2019096796A1 (en) * | 2017-11-14 | 2019-05-23 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Non-human papillomaviruses for gene delivery in vitro and in vivo |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6599739B1 (en) * | 1996-07-17 | 2003-07-29 | The United States Of America As Represented By The Department Of Health & Human Services | Infectious papillomavirus pseudoviral particles |
| KR20020084620A (en) * | 2001-05-03 | 2002-11-09 | 오유경 | Gene delivery system containing a desired gene in virus-like particles |
| CN101487009A (en) * | 2008-01-15 | 2009-07-22 | 上海泽润生物科技有限公司 | Method for preparing vaccine for anti-HPV 16 infection by pichia yeast expression system |
| CN115960178A (en) * | 2022-12-28 | 2023-04-14 | 北京康乐卫士生物技术股份有限公司 | Expression of human papilloma virus HPV59L1 protein, virus-like particle and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Virus-like Particles as Nanocarriers for Intracellular Delivery of Biomolecules and Compounds;Junyao He et. al;viruses(第14期);第1-18页 * |
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